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A tour through CATH and a story of survival
You are interested in a beta lactamase protein that you know confers drug resistance in a pathogenic bacterium and you want to find out more about the nature of the active site and why this pathogen is resistant to penicillin. Search Genome3D ( www.genome3d.eu) with the UniProt identifier P00811. You will see that the protein belongs to CATH superfamily 3.40.710.10 of beta-lactamases and DD-peptidases. The majority of Genome3D structure prediction methods assign the protein to equivalent superfamilies in SCOP and CATH.
Click on the Gene3D link to get more information from the CATH-Gene3D resource (or click here if you get lost). Click on the Drugs entry of the menu on the left of the page to see information available on the drugs found to bind to the active site of this protein. There is one approved synthetic drug and many other putative drugs for which there is experimental evidence. Go back to the top of the page and take note of the fact that P00811 is in Functional Family 22208 of beta-lactamases and that this is associated with the EC code 22.214.171.124. We will investigate Functional Family 22208 later in the tutorial.
Now let's look at the CATH superfamily (3.40.710.10). Click on CATH superfamily (or here). This page shows summary information for superfamily 3.40.710.10. Mouse-over the Species Diversity box to see the species distribution of the superfamily. You will see that this superfamily is largely confined to bacterial species. Now mouse-over the EC Diversity box to see the functions associated with different relatives in the superfamily. Apart from beta-lactamases, what is the largest functional group?
What about the evolution of the proteins?
To find out when these different functions emerged in evolution, click on the FunTree box (under Enzyme Function). You can see that FunTree provides a lot of phylogenetic and functional information for the superfamily. This is organised according to sets of similar structural groupings (SSG). Click on the link for this SSG (which is an image of a structure to the right of SSG on the left of the screen; click here if you get lost) and then select the Ancestral Character Estimation link on the left menu. This shows the earliest point in evolution at which a particular function can be detected within the domain superfamily. The beta lactamases (with EC code 126.96.36.199, as seen earlier) are clearly ancient and evolved at about the same time as the largest group of relatives in the superfamily, the DD-peptidases (EC code 188.8.131.52).
The DD-peptidases (or "D-Ala-D-Ala carboxypeptidases" in the EC diversity box, earlier) are bacterial enzymes that are also known as "penicillin binding proteins". They cross-link peptidoglycan chains to form a strong mesh-like structure in bacterial cell walls. The figure to the right describes their activity in Gram-positive bacteria.
These enzymes are serine peptidases i.e. during catalysis an acyl-enzyme intermediate is formed involving a serine residue. The use of D-amino acids by bacteria contributes to the resilience of their cell walls since proteins only contain L-amino acids and most peptidases are L-stereospecific.
About 2 billion years ago fungi evolved the ability to synthesize beta-lactam antibiotics which bind irreversibly in the active site of DD-peptidases and thus inhibit their activity. Because bacterial cell walls are constantly being broken down and remodelled by the bacteria themselves, an inability to regenerate cross-linkages rapidly results in loss of integrity of the cell wall and bacterial death. An example of a current day organism is the penicillin mould that produces penicillin and in 1928 this was the first antibiotic to be discovered by Man.
In response to this assault it is generally accepted that some bacterial DD-peptidases evolved into beta-lactamases that can break open the beta-lactam ring in an antibiotic and thus inactivate it.
How has resistance evolved? Can we see changes in the active site?
Let's look more closely at these two different relatives. Go back to the Gene3D summary page for our protein P00811. We can look at the sequence aligned to functionally similar sequences by clicking Domain Table -> see Modelled Structure).
We can also look more closely at the functional family (FunFam) cluster in CATH by clicking Domain Table -> CATH FunFam. FunFams are clusters of relatives that are very likely to have similar functions. Click on Alignments in the middle of the SUPERFAMILY LINKS menu at the top left. Find FunFam 22208 (Beta-lactamase [FF: 22208] in the table (type "22208" into the filter at the top of the table) and open the link in a new browser tab. This FunFam contains our original beta-lactamase, P00811.
Select the Alignment tab to view the multiple sequence alignment of this functional family. Highly conserved positions have been identified by the Scorecons algorithm ("Scoring residue conservation" Valdar WSJ (2002), Proteins: Structure, Function, and Genetics. 43(2): 227-241) and are shown highlighted in green. Can you spot the SXXK motif associated with the catalytic dyad used by this family? It should be around alignment positions 95-98.
Now switch back to the browser tab containing the table of FunFams, open FunFam 22165 (D-alanyl-D-alanine carboxypeptidase [FF: 22165]) in a new browser tab and click on the page's Alignment tab. FunFam 22165 contains DD-peptidase relatives. You will see a similar motif as both enzymes possess the SXXK motif catalytic dyad (additional catalytic residues are often suggested but these are also shared by both classes of enzyme). This should be around alignment positions 43-46. So the difference in their catalytic activity is likely to be due to substrate binding residues that perhaps subtly alter the position of the substrate, alter the energetic stability of binding, stabilization of the transition state intermediate and/or subsequent hydrolysis of the acyl-enzyme intermediate.
Can you see any highly conserved residues which lie close to the SXXK motifs but which are different in the beta-lactamase and DD-peptidase alignments? For example, the following positions involve quite significant changes: 100 (Phe) in 22208 versus 48 (Met) in 22165, and 103 (Val) in 22208 versus 51 (Tyr) in 22165.
How close are these residue mutations?
CATH provides two structural comparison resources. The CATHEDRAL web-server allows users to submit a structure to be scanned against a library of representative CATH domain structures. The SSAP web-server allows a quicker structural alignment and superposition of two structures.
To save time in this tutorial, we have performed these searches for you. If you click here, you'll see a search of the 1my8A00 beta-lactamase structure against CATH. The top hit is for another beta-lactamase within the same superfamily. Now let's compare the structure of the beta-lactamase with a structure from a DD-peptidase family. Click on this PyMOL superposition (which was pre-prepared using the SSAP web-server mentioned above) of protein structure 1my8A00 for the beta lactamase P00811 against the structure 3itbD01 for the DD-peptidase P08506. How similar are the structures? The residues involved in the SXXK motif in the active site are highlighted in green. How similar are the active site regions of these functionally diverse domains?
Here are some residue positions that are highly conserved within each FunFam but different between the two FunFams:
22208 (1my8A00) : ...SXXK.F..V...
22165 (3itbD01) : ...SXXK.M..Y...
You can verify that each of these is highly conserved in the two FunFam alignment browser tabs you now have open. Let's select these residues in PyMOL: look at the two sequences at the top of the PyMOL window, find the SXXK motif (highlighted in green) and then, for both sequences, select the residues two and five residues after the K. (Note: the two sequences aren't aligned in PyMOL so these residues are in different positions.) Type show sticks, sele and then look closely at the side chains of these residues. Would you say that these mutated residues are likely to have an impact on the binding of the peptide substrate?
Some thoughts to take away
Novel beta-lactamases rapidly evolve and confer resistance against new Man-made derivatives of beta-lactam antibiotics (typically within two years) as is the case for all other existing classes of medicinal antibiotics, resulting in what may be an imminent crisis for modern medicine. People may start dying from scratches again as they often did in the pre-antibiotic days of previous centuries. Pharmaceutical companies are reluctant to invest in the development of new classes of antibiotics since they would need to be kept in reserve for emergencies and thus be infrequently used and result in little revenue.